We have continued to characterize factors involved in regulating melanocyte function in the skin. The specific aims of this project include: (1) Identify and characterize factors that regulate melanocyte function; (2) Characterize trafficking mechanisms of melanosomal proteins and their disruption in pigmentary diseases; (3) Elucidate downstream effects of MC1R signaling by its agonists and antagonists; and (4) Characterize the regulation of melanocyte function by UV radiation. Regulation at the Enzymatic Level - We previously demonstrated that TYR, TRP1 (now TYRP1) and TRP2 (now DCT) have distinct catalytic functions and interact within a complex in melanosomes. TYR has the critical catalytic function in melanogenesis, i.e. the hydroxylation of tyrosine to DOPA, but TYRP1 and DCT have important post-TYR functions that modify the type of melanin synthesized. Those post-TYR reactions not only optimize the physical characteristics of melanin produced and maximize its photoprotective benefits, but they also minimize the inherent cytotoxicity of melanogenic precursors and their by-products. Mutations in the genes encoding TYRP1 or DCT often result in premature melanocyte death, presumably from cytotoxic intermediates. The sorting signals and trafficking pathways of all 3 TRPs have been actively studied. Regulation at the Structural Level - Pmel17/gp100 was recently shown to play an important role in the maturation of vesicular Stage I melanosomes to the fibrillar Stage II form. That transition is essential to the subsequent targeting and delivery of melanogenic enzymes to melanosomes and is required for melanin synthesis and deposition to occur. We showed that Pmel17 undergoes extremely complex glycosylation and can traffic via a number of distinct sorting pathways to reach melanosomes (and other subcellular compartments). Pmel17 can be delivered directly to early melanosomes via an AP-mediated sorting pathway but the remainder is sorted to the plasma membrane and can then be redirected to early melanosomes or secreted to the extracellular milieu. The involvement of polarized elements in the sorting of Pmel17 demonstrated that melanocytes are in fact polarized cells. In retrospect this is not surprising given the dual and quite distinct roles of melanocytes in the skin, attached on their lower surface to the basement membrane and on their top surface to keratinocytes. Once delivered to Stage I melanosomes, Pmel17 is cleaved at least twice, the important outcome being that the amino terminal fragment is released from the membrane and generates the internal fibrillar matrix where the melanin is deposited. Our studies have shown that the Pro-Ser-Thr rich repeat domain of Pmel17 (called RPT) is essential for the formation of fibrils; although deletion of the RPT domain does not affect Pmel17 trafficking, fibril formation is disrupted. Mutations in Pmel17 can be quite toxic and in mice the gene is called silver because follicular melanocytes die and the hair becomes prematurely gray. Regulation at the Cellular Level - Factors such as UV, DKK1, MSH and ASP, regulate the quantity and quality of melanins produced in the skin by melanocytes. We have detailed molecular changes in gene expression, and mechanisms involved in those changes, which occur in response to various physiological stimuli. The G-protein coupled melanocortin receptor 1 (MC1R) is regulated by its agonist MSH and antagonist ASP. Those interactions regulate skin and hair color, as well as responses to the environment. Our research on MC1R has involved characterizing: (1) MC1R genotype and melanocyte phenotype, (2) mechanisms involved with MC1R activation or inhibition by MSH or ASP, (3) downstream events that regulate MC1R function in determining melanocyte differentiation, and (4) the involvement of MC1R function in responses of melanocytes to other physiological stimuli, such as UV. The switch to produce yellow/red pheomelanin or black/brown eumelanin is under the control of the MC1R and its ligands, but the catalytic level at which this is modulated has remained elusive. The intramelanosomal level of cysteine is certainly an important factor and our proteomics analysis of melanosomes has identified a number of sulfhydryl regulating factors that might be involved. We reported recently that MSH induces MC1R expression by melanocytes whereas the antagonist ASP seems to have the opposite effect. Semi-quantitative real-time RT-PCR showed that MC1R transcript levels are up-regulated by MSH (4-fold) but are not significantly affected by ASP. Since MC1R is the only receptor known that binds ASP, a minimal expression level of it is required for melanocytes to respond to ASP. Further, since the MC1R switch is reversible, expression of a minimum level of MC1R is required to allow MSH to re-induce eumelanogenesis. While MSH regulates MC1R function at both the mRNA and protein levels, ASP acts only on its translation. We also characterized the influence of dermal fibroblasts on skin pigmentation. We examined whether melanocyte differentiation is regulated by factors originating from fibroblasts in the dermis. Melanocyte density in palmoplantar human skin (i.e. skin on the palms and soles) is 5X less than that found elsewhere on the body (i.e. non-palmoplantar skin). We showed that fibroblasts derived from palmoplantar skin significantly suppress the growth and pigmentation of melanocytes compared with non-palmoplantar fibroblasts. Using cDNA microArray analysis, we determined that fibroblasts from palmoplantar skin express much higher levels of DKK1, an inhibitor of canonical Wnt signaling, compared to fibroblasts derived from trunk skin. Transfection studies revealed that DKK1 dramatically inhibits melanocyte function, probably through beta-catenin-mediated regulation of MITF which in turn modulates the growth and differentiation of melanocytes. These results explain why skin on the palms and soles is hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in palmoplantar areas during embryogenesis. Treatment of keratinocytes with DKK1 increases their proliferation and decreases their melanin uptake, whereas treatment of melanocytes with DKK1 decreases their growth and differentiation. Numerous genes are regulated by DKK1 in both types of cells, expression patterns that were validated for many of the more interesting genes by RT-PCR, immunohistochemistry and/or Western blotting. We verified these effects by treating MelanoDerm skin with DKK1 which induced a thicker and less pigmented morphology and reproduced the expression patterns of key proteins observed in palmoplantar skin. In sum, these studies elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical/site-specific differences in DKK1 secretion by dermal fibroblasts which affects the overlying epidermis.